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OBSERVATIONS ON THE GROWTH OF STREP- 
TOCOCCI IN BLOOD-CARBOHYDRATE 
MEDIUM * 

David John Davis 

From the Department of Pathology and Bacteriology of the University of 
Illinois, Chicago 

Carbohydrates not only serve as an important food substance for 
many bacteria but are most useful for the identification and differen- 
tiation of closely related varieties. Blood and blood serum in recent 
years have come into use quite extensively as an aid in differentiation 
of organisms. In addition to other means the ability of germs to lake 
the 'corpuscles has been taken advantage of, especially in connection 
with the examination of cocci. The interaction, therefore, of blood 
and carbohydrates in mediums under the influence of bacterial 
growth is of significance both practically and theoretically. 

Since Schottmuller called attention to the hemolytic action of 
streptococci on blood and on this basis divided them into 3 types: 
the hemolyticus, the viridans, and the mucosus, this method has in 
many laboratories come into general use for isolating and differentiat- 
ing this group. For this purpose it has proved an invaluable aid. 
By many workers, especially in this country for the past 15 years, 
the method has been tested out quite thoroughly and its limitations 
and possibilities have been fairly well determined. I may refer in 
this regard to the early work in this country of Weaver, Ruediger, 
and Rosenow, to the more recent work of Smith and Brown, 1 and 
to the classifications of streptococci advocated by Holman 2 and by 
Lyell ; 3 also to the recent work of Becker. 4 The hemolytic property, 
like many other properties of bacteria, is not absolutely constant but 
ordinarily under uniform conditions is sufficiently so to be of real 
value in differentiation. 

It has long been known that sugar in mediums interfered with 
the production of characteristic zones of hemolysis on plate cultures. 

* Received for publication June 2, 1917. 

1 Jour. Med. Research, 1914, 31, p. 455. 

2 Ibid., 1916, 34, p. 377. 

3 Ibid., 1914, 30, p. 487. 

* Jour. Infect. Dis., 1916, 19, p. 754. 



Growth of Streptococci in Blood-Carbohydrate Medium 309 

It was pointed out by Ruediger 5 that in glucose blood mediums hemolytic 
streptococci failed to produce a clear zone, but became surrounded by an indefi- 
nite greenish or brownish zone. He showed also that lactic acid when dropped 
on a blood-agar plate caused a similar greenish zone to form about itself. 
Cole has demonstrated that pneumococci change oxyhemoglobin to methhemo- 
globin in artificial medium, and that the greenish or greenish-brown color 
about pneumococcus colonies on blood plates is due to this phenomenon. Blake' 
has made the same observation with streptococcus viridans. Sugars very appre- 
ciably accelerate this phenomenon. 

Hiss 8 called attention to the value of inulin serum water mediums in the 
differentiation of pneumococci and streptococci. As a result of the fermenta- 
tion of inulin by pneumococci, there resulted from the acids formed a precipi- 
tation and coagulation of the albuminous substances in the medium, causing 
it to become white and solid. This change does not occur in such medium 
inoculated with streptococci. This method has been modified by Ruediger," 
who used a higher proportion of serum or ascites fluid in order to facilitate 
the growth of certain strains of pneumococci, which did not grow well in 
Hiss serum water medium. 

Blood mediums should be standardized more carefully than they 
have been in the past. Attention has been called to this by nearly 
everyone who has worked on the subject. Uniform methods should 
be used so that the results of various workers may be comparable. 

Blood, when used in mediums, should be reasonably fresh. Stand- 
ing in a cool chamber for several days, however, does not impair its 
value to any marked degree. There are certain appreciable differences 
between human blood and the blood of various animals as shown by 
Becker. 4 One-half c.c. of defibrinated human blood added to 5 c.c. 
of melted agar cooled to approximately 42 C. is the proportion of 
blood used in the experiments herein reported. 

With a series of strains of streptococci, I have made observations 
on the effect of their growth on blood mediums containing a variety 
of sugars both fermentable and nonf ermentable ; 1% sugar mediums 
were first used, but on account of an occasional indefinite or delayed 
reaction the amount of sugar was increased to 2 or 3% in the 
mediums and either one of these amounts was used in the later 
experiments. I should recommend 2% for routine work. 

It was noted that when streptococci are grown on fermentable 
sugar blood mediums, the mediums acquire a brown turbidity, begin- 
ning at first near the colonies, and later involving the entire plate or 
tube. This change may be attributed to the action of acids produced 

5 Jour. Infect. Dis., 1906, 3, p. 663. 

6 Jour. Exper. Med., 1914, 20, p. 363. 

7 Ibid., 1916, 24, p. 315. 

8 Ibid., 1902, 10, p. 317. 

9 Jour. Am. Med. Assn., 1906, 47, p. 1171. 



310 



David John Davis 



by the bacteria, and in order to analyze this point further systematic 
tests have been made of the affects of the growth of a number of 
strains of streptococci on mediums containing the various sugars. 
The results are given in Table 1. 

This table shows the fermentation of sugars as determined by 
titration, and it also indicates by the plus sign those blood-sugar plates 
and tubes which developed the striking brown turbidity. It will be 
seen that the reaction as manifested by the development of brown 
turbidity corresponds in every instance to the positive fermentation 
tests. With those sugars which are not fermented by the streptococci 
the blood mediums remain red, and the typical clear zones of hemolysis 
on plate cultures are quite like those produced on plain blood-agar 
plates. 

TABLE 1 

Effect of Streptococci when Grown on Carbohydrate (2%) Blodd Agar Plates and Tubes 





Glucose 


Lactose 


Maltose 


Salicin 


Number of 
Strain 










Blood 


Titra- 


Blood 


Titra- 


Blood 


Titra- 


Blood 


Titra- 




Reaction 


tion 


Reaction 


tion 


Reaction 


tion 


Reaction 


tion 


72 


+ 


6.05 


+ 


4.37 


+ 


5.05 


+ 


6.05 


183 


+ 


5.15 


+ 


5.05 


+ 


5.05 


+ 


5.05 


30O 


+ 


6.25 


+ 


4.55 


+ 


5.55 


+ 


5.05 


228 


+ 


6.35 


+ 


2.71 


+ 


5.25 


+ 


4.71 


140 


+ 


5.87 


+ 


4.55 


+ 


5.05 


+ 


4.85 


290 


+ 


5.05 


+ 


4.65 


+ 


3.35 


+ 


5.05 


41 


4 


6.55 


+ 


S.65 


+ 


5.25 


+ 


4.05 


134 


+ 


6.37 


+ 


4.55 


+ 


4.05 


+ 


4.55 


310 


+ 


6.05 


+ 


4.87 


+ 


5.05 


+ 


5.35 


6 


+ 


6.10 


+ 


4.60 


+ 


5.30 


+ 


5.10 


211 


+ 


4.55 


+ 


3.65 


+ 


3.85 


+ 


4.35 



These reactions are so clear cut that sugar blood tubes may be 
used for determining fermentation properties instead of litmus 
mediums. By adding a small amount of lactic acid to mediums, either 
in plate or tube, the change that occurs is quite indistinguishable 
from that caused by the growth of the acid-producing bacteria. 

The zone of hemolysis on the fermentable sugar-blood plates was 
often indistinct, and at times not visible at all. Sometimes the zone 
at first was fairly clear, later becoming more turbid, but not persisting 
as a clear and definite zone, like that which hemolytic streptococci 
produce on the plain blood mediums. 

This reaction is entirely comparable to the reaction observed in 
the sheep serum inulin mediums devised by Hiss for the differentia- 
tion of streptococci and pneumococci. When the defibrinated blood 
is used we get not only a precipitation of the serum constituents, but 
also a brownish discoloration of the red blood cells, due to acids and 
no doubt other changes which make the differentiation decidedly 



Growth of Streptococci in Blood-Carbohydrate Medium 311 



striking. Since blood and carbohydrates are no doubt coming more 
and more into use in growing and determining bacteria, I suggest this 
method as of importance in the study and classification of members 
of the pneumo-streptococcus group. 

An attempt was made to test the relation of hemolysis to acid- 
production by streptococci. An experiment was designed to neutral- 
ize the effect of the acid by the addition of powdered CaCO s to the 
blood-sugar mediums. 

Glucose and lactose were used. On such mediums when freshly made and 
plated the particles of CaCOs are seen evenly distributed. After incubation 
the CaCOs particles around the streptococcus colonies are dissolved, to some 
extent in 24 hours, much more so in 48 or more hours. The zone of solu- 
tion of CaC0 3 particles is larger than the zone of hemolysis. For 24-48 hours 

TABLE 1. — Continued 
Effect of Streptococci when Grown on Carbohydrate (2%) Blood Agar Plates and Tubes 



Saccharose 


Mannite 


Rafflnose 


Inulin 


Blood 


Titra- 


Blood 


Titra- 


Blood 


Titra- 


Blood 


Titra- 


Reaction 


tion 


Reaction 


tion 


Reaction 


tion 


Reaction 


tion 


+ 


Acid 


-t- 


3.35 





1 





0.95 


+ 


Acid 





1.00 





1 





0.95 





1.5 





1.00 





1.1 





1.25 


+ 


4.65 





1.5 





1.5 





1 


+ 


3.55 


+ 


3.05 





1 





0.91 


+ 


5.05 





0.95 





0.83 





1.2 


+ 


4.25 





0.95 





1 





1 


+ 


4.6 





0.98 





0.85 





1.08 


+ 


5.55 





0.9 





1.2 





0.91 


+ 


4.70 





1 





0.9 





1.08 


+ 


3.55 





0.9 





1 





1.08 



the blood on the CaCOs plates does not change materially; the carbonate seems 
to be appreciably protective for the corpuscles for a time at least. Later the 
plates slowly turn brownish and turbid, but usually not to the same extent 
as in the plates without CaCOs. The colonies on the CaCOs plates become 
somewhat larger than when grown on mediums without CaCOs. 

Control experiments were made to test the effect of CaCOs on plain blood- 
agar plates and the streptococcus hemolytic zones. The plain blood agar con- 
taining CaC0 3 remains red and appears fresh for a week or more, at room 
or incubator temperature, and shows no difference from a control plate made 
without CaCOs. On CaC0 3 blood plain agar plates sown with hemolytic strep- 
tococci, the zones are about normal in size and appearance and remain so for 
a week or longer. About the colonies on such plates the particles of CaCOs 
remain indefinitely. Therefore the zone of hemolysis is not affected by CaCOs, 
and it would seem further that it bears no relation to acid-production in the 
case of typical hemolytic streptococci. Twenty strains were tested and all 
reacted alike. 

The question as to whether sugars inhibit hemolysis was inquired into, 
using the blood-agar plate. Six typical hemolytic streptococci were tested first 
on plain blood plates to determine the diameter of the zone of hemolysis. 
This was found to vary 2-4 mm. for the various strains. They were also 



312 David John Davis 

plated on CaCOs blood-glucose agar, and the size of the zones were noted 
and compared with the controls. The average size of the zones of many 
colonies of each of the strains revealed no essential differences. It was noted 
here again that the CaC0 3 particles in the colony, and for some distance about 
it, were dissolved in the sugar mediums. The clearing zone of the CaCOa 
particles is decidedly larger than that of the blood corpuscles. This is another 
point indicating that the zone of hemolysis is not dependent on acid-production. 
That acid- and hemolysin-production go on together was shown by these 
experiments, but it was noted that apparently the rate of the acid-production 
is relatively slower than that of hemolysis. At any rate the blood corpuscles 
seem more sensitive to hemolysin than do the particles of CaC0 3 to the acid 
in the concentration in which these substances exist about the colonies. 

It would appear that the acid is decidedly more diffusible in the 
mediums than is the hemolysin. The hemolysin diffuses into the 
medium rather rapidly during the first 24 hours, then slowly as judged 
from the size of the zone. Furthermore, the margin, in most strains 
of streptococci but not in all, is very clear cut and definite. On the 
sugar plates the acid diffuses rather rapidly, and continues to diffuse 
for several days so that, as a rule, sooner or later the entire plate 
becomes turbid and brown, even though few colonies may be found 
on it. The margin of the acid zone also is not clear and definite as is 
usually the margin of the zone of hemolysis. 

Since mediums are commonly made with meat or extract which 
contains some muscle sugar, the point whether or not such sugar has 
any appreciable effect on plate hemolysis was inquired into. Eight 
typical hemolyzing strains of streptococci were selected for the tests 
Plain agar was prepared, using meat; also the same medium, using 
ordinary meat extract. As controls sugar-free agar was used. On 
all these mediums no differences were noted between the character 
of the hemolytic zones about the streptococci. It would seem that the 
small amount of sugar in such mediums is too small to appreciably 
affect the nature or intensity of hemolysis. 

Attention is called to the fact that certain peptones, on the market 
especially since the war, contain considerable quantities of sugar, and 
when such are used in the preparation of mediums, they are not suit- 
able for the differentiation of bacteria by blood-agar methods. When 
Witte's peptone is not available the peptone used should be investi- 
gated as to its sugar content. This point is especially important in the 
preparation of standard medium and particularly of sugar-free 
medium. I would point out also that certain peptones darken the 
medium to such an extent that clear cut blood reactions are not readily 
obtained. 



Growth of Streptococci in Blood-Carbohydrate Medium 313 

The amount of NaCl in medium is important in relation to hemol- 
ysis. When no salt is added, as is well known, blood introduced into 
such mediums immediately hemolyzes. Plain agar was prepared con- 
taining amounts of NaCl, 0.1%-1.5%. Ten strains of hemolytic strep- 
tococci, 5 virulent and 5 nonvirulent, were tested on this medium to 
which one-third c.c. of human defibrinated blood to 5 c.c. was added 
before plating. The fragility of the corpuscles in this medium is a 
little below the resistance in pure salt solution, as might be expected. 
At 0.4%, at which in pure salt solution there is slight hemolysis, there 
was apparently no hemolysis as observed both with the microscope and 
the naked eye. Above this point the zones of hemolysis were normal 
in the various concentrations tested. If to such salft-free medium or 
medium with low NaCl content, sugar, either fermentable or nonfer- 
mentable, be added in the usual concentration of 1 % or in any sufficient 
concentration, hemolysis will not take place, because the sugar protects 
the corpuscles. For such medium, therefore, salt is not a necessary 
constituent for the prevention of hemolysis. 

SUMMARY 

Streptococci when grown on a fermentable sugar-blood medium 
cause a characteristic brownish turbidity which soon involves the 
entire tube or plate. 

On nonfermentable sugar-blood medium no such change is noticed, 
hemolysis occurring as on plain blood mediums. 

This change is sufficiently definite and constant to be of value 
in determining carbohydrate reactions. 

It is useful in routine work since practically all varieties of strep- 
tococci grow well in this medium. 

Powdered CaCO s added to sugar-blood agar will protect the 
medium for a time against the acids formed, but not permanently. 

The carbonate added to plain blood medium has no appreciable 
effect on hemolysis by streptococci. 

The presence of sugar in various peptones, at present on the 
market, is a disturbing factor in the blood-plate culture of bacteria.